Negative selection and regulatory T cell development in the thymus are critical components of central and peripheral tolerance. Both of these processes have been linked to the affinity of the TCR for self-peptide:MHC complexes; positive selection requires weak interactions with such self-antigens while natural Treg development and negative selection typically require moderate to higher affinity interactions with such antigens. We now have a substantial albeit incomplete knowledge base about the affinity and avidity of peptide ligands involved in these processes. In contrast, the basic signaling pathways that underlie negative selection and Treg development still remain poorly established. While individual signaling molecules that regulate negative selection and Treg development have been identified, we lack a basic understanding of the larger pathways involved in these processes and how similar signaling pathways can give rise to diametrically opposed outcomes - namely death of a thymocyte via negative selection versus survival via differentiation into a Treg. The central hypothesis underlying this grant is that these processes are regulated by three members of the tumor necrosis receptor superfamily (TNFRSF) - GITR, OX40 and TNFR2. Our preliminary data document that expression of these three TNFRSF receptors correlates directly with TCR signal strength and is required for Treg development. We propose that TCR signal strength is manifested as increasing expression of specific TNFRSF members and that higher TNFRSF signaling leads to preferential development of Tregs, thereby skewing the Treg TCR repertoire in favor of higher self-reactivity. Additional preliminary data suggest that GITR, OX40 and TNFR2 also influence TCR-dependent negative selection. We propose that signaling via GITR, OX40 and TNFR2 primes developing thymocytes for both negative selection and Treg development via distinct downstream signaling pathways. In our model TNFRSF signaling primes CD4+ thymocytes for cell death as well as cell responsiveness to IL2; those cells that subsequently receive an IL2 signal preferentially differentiate into Tregs while those that fail to initiate IL2-dependent signals undergo clonal deletion. The studies proposed in this grant will directly test these hypotheses as well define how the costimulatory receptor CD28 impinges on these processes. Finally, we will determine whether altering TNFRSF-dependent signals specifically in Tregs alters their function in a model of graft-versus-host disease. Thus, the studies proposed in this grant application will elucidate how specific TNFRSF members link TCR and CD28 signals to the establishment of a functional Treg pool and help broaden the repertoire of thymocytes undergoing negative selection. These studies will also illuminate how TNFRSF agonists and antagonists could be used to augment the therapeutic potential of Tregs in the future.